Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSmetabolite production, hyphal morphology, conidiation, and pellet formation [1]. Here we describe the characterization of PcVelB, PcVelC, and PcVosA asnovel subunits of this velvet complex. Using yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC), we demonstrate that allvelvet proteins are part of an interaction network. Functional analyses using single and double knockout strains generated by the FLP/FRT recombinationsystem [2] clearly indicate that velvet subunits have opposing roles in the regulation of penicillin biosynthesis and light-dependent conidiation. Moststrikingly, a direct interaction of PcVelB with an enzyme of the penicillin biosynthesis pathway, the isopenicillin N synthase was identified during yeast twohybridanalysis with PcVelB as bait. This surprising interaction was confirmed with BiFC in vivo, thereby localizing the interaction in dot-like structures inthe cytoplasm. Our discovery of a direct interaction of the isopenicillin N synthase with a subunit of the velvet complex implies a novel regulatorymechanism how enzymes of penicillin biosynthesis are regulated at the molecular level. The results provided here contribute to our fundamentalunderstanding of the function of velvet subunits as part of a regulatory network mediating signals responsible for morphology and secondary metabolism,and will be instrumental in generating mutants with newly derived properties that are relevant to strain improvement programs.[1] Hoff B, Kamerewerd J, Sigl C, Mitterbauer R, Zadra I, Kürnsteiner H, Kück U (2010) Eukaryot Cell: 9:1236-50[2] Kopke K, Hoff B, Kück U (2010) Appl Environ Microbiol 76:4664-4674.50. Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases. Gerald Lackner, Mathias Misiek,Jana Braesel, Dirk Hoffmeister. Pharmaceutical Biology, Friedrich-Schiller-University Jena, Germany.Polyketide biosynthesis is a rich source of pharmaceutically active secondary metabolites present in fungi. Besides lipid-lowering drug lovastatin, manyinfamous toxins are produced via this pathway. While abundant in Aspergillus, only few polyketides have been isolated from basidiomycetes. Highthroughput genome sequencing projects, however, now help estimate the genetic capacity of basidiomycetes to biosynthesize polyketide derivatives. Byinspection of 35 sequenced basidiomycete genomes we identified and annotated 111 iterative type I and three type III polyketide synthase (PKS) genes.Phylogenetic analyses of KS genes imply that all main families of fungal PKS had already evolved before the Ascomycota and Basidiomycota diverged. Acomparison of genomic data and metabolomic records shows that the number of polyketide genes surpasses the number of known polyketidesconsiderably. This work might serve as a guide for upcoming genomic mining projects to discover novel polyketide derivates from mushrooms.51. Engineering Cyclic Peptide Biosynthesis in Poisonous Mushrooms. Hong Luo, John S. Scott Craig, Robert M. Sgambelluri, Sung-Yong Hong, Jonathan D.Walton. Department of Energy Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, United States.Ninety percent of fatal mushroom poisonings are caused by alpha-amanitin and related bicyclic peptides found in some species of Amanita, Galerina,Lepiota, and Conocybe. We showed that the amatoxins (mainly amanitins) and related phallotoxins are synthesized on ribosomes in A. bisporigera and theunrelated mushroom G. marginata. The primary gene products are short (34-35 amino acid) proproteins that are initially processed by a dedicated prolyloligopeptidase. A genome survey sequence of A. bisporigera suggested that it has a repertoire of over 40 cyclic peptides, all produced on a singlebiosynthetic scaffold. Members of this extended gene family are characterized by conserved upstream and downstream amino acid sequences, includingtwo invariant proline residues, flanking a six to ten-amino acid “hypervariable” region that encodes the amino acids found in the mature toxins (orpredicted toxins). The evidence indicates that A. bisporigera has evolved a combinatorial strategy that could in principle biosynthesize billions of smallcyclic peptides. In order to study the other steps in amanitin biosynthesis, and to engineer novel cyclic peptides, we have developed a transformationstrategy for the amanitin-producing mushroom G. marginata. This first transformation method uses Agrobacterium-mediated transformation followed byhygromycin selection. Taking advantage of this platform, we are introducing artificial toxin genes that are deliberately designed to provide insights into thepathway. The synthetic genes include those that encode the cyclic octapeptide beta-amanitin, the heptapeptides phalloidin and phallacidin, examples ofthe toxin gene family known from A. bisporigera but not G. marginata, and randomly generated artificial sequences. Currently, thousands of transformantshave been generated through an efficient pipeline and the transformants are being analyzed for production of the expected products. If successful, thenovel peptides will be screened in a number of assays including RNA polymerase (the site of action of alpha-amanitin), membrane ion channels,pathogenic bacteria, and cancer cell lines.52. Spatial assessment of oxidative and enzymatic reactions in brown rotted wood. Jon R. Menke 1 , Jae San Ryu 2,5,6 , Gerald N. Presley 1 , Shona M. Duncan 1 ,Joel A. Jurgens 3 , Robert A. Blanchette 3 , Timothy R. Filley 4 , Kenneth E. Hammel 2,5 , Jonathan S. Schilling 1 . 1) Department of Bioproducts and BiosystemsEngineering, University of Minnesota, St. Paul, MN; 2) Department of Bacteriology, University of Wisconsin, Madison, WI; 3) Department of PlantPathology, University of Minnesota, St. Paul, MN; 4) Department of Earth and Atmospheric Sciences and the Purdue Climate Change Research Center,Purdue University, West Lafayette, IN; 5) Institute for Microbial and Biochemical Technology, U.S. Forest Products Laboratory, Madison, WI; 6) Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Republic of Korea.Brown rot fungi are theorized to use coordinated free radical oxidations and enzymatic reactions to consume wood. Though likely incompatible in vitro, itis proposed these reactions occur concurrently during brown rot of wood. We mapped and then compared fungal growth, wood modifications related tonon-enzymatic mechanisms, and cellulase activity in thin spruce ‘wafers’ to investigate the degree of spatial coincidence of these reactions in wooddegraded by Postia placenta. Nearly coincident oxidative and enzymatic reaction fronts were observed behind the most advanced hyphal tips, suggesting afine-scale (likely sub-micron) spatial or biochemical extracellular mechanism may protect both hyphae and enzymes from oxidative stress. To furtherinvestigate a possible role of enzymatic reactions in the primary depolymerization of lignocellulose during brown rot, we have initiated a study totemporally assess the depth of penetration of an endoglucanase into wood cells during this process. Previous studies have used the marker proteinsinsulin (5.7 kDa), myoglobin (17.6 kDa), and ovalbumin (44.4 kDa); and immunocytochemical electron microscopy to demonstrate the ability of the twosmaller proteins to infiltrate the cell walls of rotted wood. The Postia placenta endoglucanase Cel5B (PpCel5B) has a theoretical molecular weight of 34.6kDa, which is considerably lower than the molecular weight of ovalbumin. Our approach involves using a polyclonal antibody raised against PpCel5Bheterologously expressed in Pichia pastorius. This antibody will be used to assess the extent to which a native brown rot endoglucanase is able topenetrate Pinus resinosa cells. Given the common supposition that pore size prevents brown rot fungal endoglucanases from accessing wood secondarywalls, even in late decay stages, this work will provide a direct assessment of enzyme ingress.53. Molecular biological basis for statin resistance in naturally statin-producing organisms. Ana Rems, Rasmus Frandsen. DTU Systems Biology, TechnicalUniversity of Denmark, Kongens Lyngby, Denmark.Secondary metabolites can be toxic to the organism producing them; therefore gene clusters for biosynthesis of secondary metabolites often includegenes responsible for the organism’s self-resistance to the toxic compounds. One such gene cluster is the compactin (ML-236B) cluster in Penicilliumsolitum. Compactin is an inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, and is used as a precursor for production of the cholesterolloweringdrug pravastatin. The compactin gene cluster includes two genes encoding proteins that may confer the self-resistance to compactin and its134